The broad goal of this project is to improve the clinical diagnosis of infectious disease by advancing the current state of serological testing. Despite major advances in research techniques in microbiology, routine clinical diagnosis of infectious diseases is still lacking in many respects: infections are commonly unidentified or misidentified, the identification is too slow for optimal selection of treatment, or testing is too expensive and labor-intensive to perform on a regular basis. Our goal is to create new diagnostic capabilities that will enable a paradigm shift from iterative testing to single-step identification of a broad set of infectious agents presenting with similar symptoms. To accomplish this goal, we propose a multiplexed, array-based approach, allowing simultaneous, rapid, convenient, and sensitive measurements. The final product will be a clinical laboratory system and assay kits for multiplexed serology, providing the clinical community with a practical and cost- effective analytical approach to aid in the diagnosis of infectious diseases. This application is focused on the diagnosis of encephalitis, which could be significantly aided by multiplexed serology testing. Encephalitis is an acute inflammatory process affecting the brain that has many potential causes. It is a complex and often severe syndrome causing significant morbidity and mortality, and unfortunately, the etiology is not identified in most cases (Griffin 2005, Bloch 2007). In Phase 1 we will target the detection of 7 common causes of viral and bacterial encephalitis in the U.S.: West Nile virus (WN), St. Louis encephalitis virus (SLE), La Crosse encephalitis virus (LAC), Western and Eastern equine encephalitis viruses (WEE and EEE), Lyme disease caused by the bacteria Borrelia burgdorferi, and cat scratch disease caused by the bacteria Bartonella henselae. In Phase 2, we will expand this panel to include additional causes of encephalitis. We will also automate the testing by adding sample handling and assay processing capabilities to our existing research-use instrumentation. Beyond Phase 2, we intend to take the proposed system and assays through regulatory approval/clearance by the FDA. To perform multiplexed serology assays, we will use MSD's Multi-Array platform, which is designed for measuring multiple analytes in each well of a 96-well plate. We will pattern viral/bacterial antigen arrays in each well, and independently measure both IgG and IgM antibodies to each antigen, to aid in determining the phase of the infection. Several innovative approaches, such as use of sensitive ECL array-based detection, extension of the typical assay dynamic range, and inclusion of multiple control assays to identify false positives and negatives due to interferences, will further enhance the utility and performance of the panel. Briefly, the specific aims are to 1) Demonstrate performance improvement of individual assays over current methods, 2) Combine assays for 7 common causes of encephalitis into a single multiplexed serology panel along with control assays, and 3) Evaluate the clinical utility of the developed methods for diagnosing the etiology of encephalitis with ~100 retrospective clinical samples (serum and CSF) from the New York State Department of Health sample repository. PUBLIC HEALTH RELEVANCE: Clinical tests will be developed to allow faster, easier and more effective diagnosis of bacterial and viral infections of the brain (encephalitis). Tests for many different causes of infection will be performed at the same time, using more sensitive and efficient measuring techniques than currently used. The tests will be more cost- effective than current methods, and will provide better and faster diagnosis and subsequent selection of treatment, thereby improving patient health care. [unreadable] [unreadable] [unreadable]